Naturally occurring trichothecenes are a group of toxic secondary fungal metabolites which are produced by a number of fungi in the general Fusarium, Trichoderma, Myrothecium and Stachybotrys. Some natural and some man made examples of trichothecenes are described in FIGS. 1 and 2. As shown in FIG. 1 of the attached drawings, trichothecenes contain a common skeleton with different side chain residues at points R.sub.1 -R.sub.5. Three typical classes of natural trichothecenes are
type "A" (where R.sub.5 is H or ISV), type "B" where R.sub.5 is .dbd.O, and type "C" where R.sub.2 is linked to R.sub.3 by a carbon chain.
Because of their toxic effects and the frequent contamination of these toxins in foods and feeds, trichothecenes are potentially hazardous to human and animal health. There is also concern regarding the possible use of these compounds as biological warfare compounds (e.g. in "yellow rain"). A dependable, sensitive, specific, simple, and inexpensive method for the detection of mycotoxins in foods, feeds, and biological fluids is therefore highly desirable. Testing for trichothecenes has in the past been extremely difficult because this group of mycotoxins does not possess a chromophore group. Methods such as thin-layer chromatography, gas liquid chromatography, and mass spectrometry, which have been used for analysis of this group of mycotoxins are lacking in sensitivity and/or specificity, or need expensive instruments. Also, extensive cleanup is generally necessary before actual analysis.
To overcome some of the difficulties encountered with these prior assay methods, attempts have been made to develop immunoassays for trichothecenes by utilizing the principle of specific antigen-antibody interaction. However, trichothecenes are small molecular weight organic compounds which are too small to lead to desired levels of antibody development. Thus, they must first be conjugated to a protein or a polypeptide carrier in order for antibodies to be developed to them. Since natural trichothecenes do not have a reactive group that can be directly conjugated to protein, it is necessary to first introduce a reactive group before the coupling reaction takes place. Approaches which have been used for conjugation involve the introduction of various reactive groups at R.sub.1 and R.sub.5.
Such studies have led to the development of specific antibodies against T-2, DAS, and DOVE. See FIG. 2 and F. Chu et al., 37 Appl. Environ. Microbiol. 104-108 (1979); G. Zhang et al., 51 Appl. Environ. Microbiol. 132-137 (1986); R. Wei et al., 49 J. Fd. Prot. 267-271 (1986); F. Chu et al., 48 Appl. Environ. Microbiol. 777-80, 781-84 (1984). The disclosure of these articles and all other articles recited herein are incorporated herein by reference as if fully set forth herein.
This approach has problems for trichothecenes such as DON, T-2 tetraol, and nivalenol. This is due to the presence of many hydroxyl groups at the R.sub.1 -R.sub.5 positions. Such groups not only create problems in conjugation to proteins, but also appear to render the molecule less immunogenic (or may produce antibodies that are currently undetectable).
Thus, it can be seen that a need exists for an improved immunoassay for trichothecenes which are of the type where at least three of the R.sub.1 -R.sub.5 moieties are OH groups.